RESUMEN
BACKGROUND: HER2 is overexpressed in 25-30% of breast cancer. Multiple domains targeting of a receptor can have synergistic/additive therapeutic effects. METHODS: Two domain-specific ADCs trastuzumab-PEG6-DM1 (domain IV) and pertuzumab-PEG6-DM1 (domain II) were developed, characterised and radiolabeled to obtain [89Zr]Zr-trastuzumab-PEG6-DM1 and [67Cu]Cu-pertuzumab-PEG6-DM1 to study their in vitro (binding assay, internalisation and cytotoxicity) and in vivo (pharmacokinetics, biodistribution and immunoPET/SPECT imaging) characteristics. RESULTS: The ADCs had an average drug-to-antibody ratio of 3. Trastuzumab did not compete with [67Cu]Cu-pertuzumab-PEG6-DM1 for binding to HER2. The highest antibody internalisation was observed with the combination of ADCs in BT-474 cells compared with single antibodies or ADCs. The combination of the two ADCs had the lowest IC50 compared with treatment using the single ADCs or controls. Pharmacokinetics showed biphasic half-lives with fast distribution and slow elimination, and an AUC that was five-fold higher for [89Zr]Zr-trastuzumab-PEG6-DM1 compared with [67Cu]Cu-pertuzumab-PEG6-DM1. Tumour uptake of [89Zr]Zr-trastuzumab-PEG6-DM1 was 51.3 ± 17.3% IA/g (BT-474), and 12.9 ± 2.1% IA/g (JIMT-1) which was similarly to [67Cu]Cu-pertuzumab-PEG6-DM1. Mice pre-blocked with pertuzumab had [89Zr]Zr-trastuzumab-PEG6-DM1 tumour uptakes of 66.3 ± 33.9% IA/g (BT-474) and 25.3 ± 4.9% IA/g (JIMT-1) at 120 h p.i. CONCLUSION: Using these biologics simultaneously as biparatopic theranostic agents has additive benefits.
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Neoplasias , Medicina de Precisión , Animales , Ratones , Distribución Tisular , Receptor ErbB-2/metabolismo , Trastuzumab/uso terapéutico , Neoplasias/tratamiento farmacológicoRESUMEN
Identification of genetic mutations in Parkinson's disease (PD) promulgates the genetic nature of disease susceptibility. Resilience-associated genes being unknown till date, the normal genetic makeup of an individual may be determinative too. Our earlier studies comparing the substantia nigra (SN) and striatum of C57BL/6J, CD-1 mice, and their F1-crossbreds demonstrated the neuroprotective role of admixing against the neurotoxin MPTP. Furthermore, the differences in levels of mitochondrial fission/fusion proteins in the SN of parent strains imply effects on mitochondrial biogenesis. Our present investigations suggest that the baseline levels of apoptotic factors Bcl-2, Bax, and AIF differ across the three strains and are differentially altered in SN following MPTP administration. The reduction in complex-I levels exclusively in MPTP-injected C57BL/6J reiterates mitochondrial involvement in PD pathogenesis. The MPTP-induced increase in complex-IV, in the nigra of both parent strains, may be compensatory in nature. The ultrastructural evaluation showed fairly preserved mitochondria in the dopaminergic neurons of CD-1 and F1-crossbreds. However, in CD-1, the endoplasmic reticulum demonstrated distinct luminal enlargement, bordering onto ballooning, suggesting proteinopathy as a possible initial trigger.The increase in α-synuclein in the pars reticulata of crossbreds suggests a supportive role for this output nucleus in compensating for the lost function of pars compacta. Alternatively, since α-synuclein over-expression occurs in different brain regions in PD, the α-synuclein increase here may suggest a similar pathogenic outcome. Further understanding is required to resolve this biological contraption. Nevertheless, admixing reduces the risk to MPTP by favoring anti-apoptotic consequences. Similar neuroprotection may be envisaged in the admixed populace of Anglo-Indians.
Asunto(s)
Intoxicación por MPTP , Enfermedad de Parkinson , Animales , Ratones , Neurotoxinas/metabolismo , alfa-Sinucleína/metabolismo , Ratones Endogámicos C57BL , Sustancia Negra/patología , Enfermedad de Parkinson/patología , Neuronas Dopaminérgicas/metabolismo , Mitocondrias/metabolismo , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Intoxicación por MPTP/metabolismoRESUMEN
Antibodies that recognize cancer biomarkers, such as MUC16, can be used as vehicles to deliver contrast agents (imaging) or cytotoxic payloads (therapy) to the site of tumors. MUC16 is overexpressed in 80% of epithelial ovarian cancer (EOC) and 65% of pancreatic ductal adenocarcinomas (PDAC), where effective 'theranostic' probes are much needed. This work aims to develop fully human antibodies against MUC16 and evaluate them as potential immuno-PET imaging probes for detecting ovarian and pancreatic cancers. We developed a fully human monoclonal antibody, M16Ab, against MUC16 using phage display. M16Ab was conjugated with p-SCN-Bn-DFO and radiolabeled with 89Zr. 89Zr-DFO-M16Ab was then evaluated for binding specificity and affinity using flow cytometry. In vivo evaluation of 89Zr-DFO-M16Ab was performed by microPET/CT imaging at different time points at 24−120 h post injection (p.i.) and ex vivo biodistribution studies in mice bearing MUC16-expressing OVCAR3, SKOV3 (ovarian) and SW1990 (pancreatic) xenografts. 89Zr-DFO-M16Ab bound specifically to MUC16-expressing cancer cells with an EC50 of 10nM. 89Zr-DFO-M16Ab was stable in serum and showed specific uptake and retention in tumor xenografts even after 120 h p.i. (microPET/CT) with tumor-to-blood ratios > 43 for the SW1990 xenograft. Specific tumor uptake was observed for SW1990/OVCAR3 xenografts but not in MUC16-negative SKOV3 xenografts. Pharmacokinetic study shows a relatively short distribution (t1/2α) and elimination half-life (t1/2ß) of 4.4 h and 99 h, respectively. In summary, 89Zr-DFO-M16Ab is an effective non-invasive imaging probe for ovarian and pancreatic cancers and shows promise for further development of theranostic radiopharmaceuticals.
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Matuzumab and nimotuzumab are anti-EGFR monoclonal antibodies that bind to different epitopes of domain III of EGFR. We developed 89Zr-matuzumab as a PET probe for diagnosis/monitoring of response to treatment of a noncompeting anti-EGFR nimotuzumab antibody drug conjugate (ADC) using mouse colorectal cancer (CRC) xenografts. We developed 89Zr-matuzumab and performed quality control in EGFR-positive DLD-1 cells. The KD of matuzumab, DFO-matuzumab and 89Zr-matuzumab in DLD-1 cells was 5.9, 6.2 and 3 nM, respectively. A competitive radioligand binding assay showed that 89Zr-matuzumab and nimotuzumab bound to noncompeting epitopes of EGFR. MicroPET/CT imaging and biodistribution of 89Zr-matuzumab in mice bearing EGFR-positive xenografts (HT29, DLD-1 and MDA-MB-231) showed high uptake that was blocked with pre-dosing with matuzumab but not with the noncompeting binder nimotuzumab. We evaluated nimotuzumab-PEG6-DM1 ADC in CRC cells. IC50 of nimotuzumab-PEG6-DM1 in SNU-C2B, DLD-1 and SW620 cells was dependent on EGFR density and was up to five-fold lower than that of naked nimotuzumab. Mice bearing the SNU-C2B xenograft were treated using three 15 mg/kg doses of nimotuzumab-PEG6-DM1, and 89Zr-matuzumab microPET/CT was used to monitor the response to treatment. Treatment resulted in complete remission of the SNU-C2B tumor in 2/3 mice. Matuzumab and nimotuzumab are noncompeting and can be used simultaneously.
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Glycogen synthase kinase 3-beta (GSK3ß) is a critical regulator of several cellular pathways involved in neurodevelopment and neuroplasticity and as such is a potential focus for the discovery of new neurotherapeutics toward the treatment of neuropsychiatric and neurodegenerative diseases. The majority of efforts to develop inhibitors of GSK3ß have been focused on developing small molecule inhibitors that compete with adenosine triphosphate (ATP) through direct interaction with the ATP binding site. This strategy has presented selectivity challenges due to the evolutionary conservation of this domain within the kinome. The disrupted in schizophrenia 1 (DISC1) protein has previously been shown to bind and inhibit GSK3ß activity. Here, we report the characterization of a 44-mer peptide derived from human DISC1 (hDISCtide) that is sufficient to both bind and inhibit GSK3ß in a noncompetitive mode distinct from classical ATP competitive inhibitors. Based on multiple independent biochemical and biophysical assays, we propose that hDISCtide interacts at two distinct regions of GSK3ß: an inhibitory region that partially overlaps with the binding site of FRATide, a well-known GSK3ß binding peptide, and a specific binding region that is unique to hDISCtide. Taken together, our findings present a novel avenue for developing a peptide-based selective inhibitor of GSK3ß.